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ABSTRACT: Microsporidia spore surface proteins are an important, under investigated aspect of spore/host cell attachment and infection. For comparison analysis of surface proteins, we required an antibody control specific for an intracellular protein. An endoplasmic reticulum-associated heat shock protein 70 family member (Hsp70; ECU02_0100; "C1") was chosen for further analysis. DNA encoding the C1 hsp70 was amplified, cloned and used to heterologously express the C1 Hsp70 protein, and specific antiserum was generated. Two-dimensional Western blotting analysis showed that the purified antibodies were monospecific. Immunoelectron microscopy of developing and mature E. cuniculi spores revealed that the protein localized to internal structures and not to the spore surface. In spore adherence inhibition assays, the anti-C1 antibodies did not inhibit spore adherence to host cell surfaces, whereas antibodies to a known surface adhesin (EnP1) did so. In future studies, the antibodies to the 'C1' Hsp70 will be used to delineate spore surface protein expression.
International Journal of Microbiology 01/2010; 2010.
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ABSTRACT: Microsporidia are spore-forming fungal pathogens that require the intracellular environment of host cells for propagation. We have shown that spores of the genus Encephalitozoon adhere to host cell surface glycosaminoglycans (GAGs) in vitro and that this adherence serves to modulate the infection process. In this study, a spore wall protein (EnP1; Encephalitozoon cuniculi ECU01_0820) from E. cuniculi and Encephalitozoon intestinalis is found to interact with the host cell surface. Analysis of the amino acid sequence reveals multiple heparin-binding motifs, which are known to interact with extracellular matrices. Both recombinant EnP1 protein and purified EnP1 antibody inhibit spore adherence, resulting in decreased host cell infection. Furthermore, when the N-terminal heparin-binding motif is deleted by site-directed mutagenesis, inhibition of adherence is ablated. Our transmission immunoelectron microscopy reveals that EnP1 is embedded in the microsporidial endospore and exospore and is found in high abundance in the polar sac/anchoring disk region, an area from which the everting polar tube is released. Finally, by using a host cell binding assay, EnP1 is shown to bind host cell surfaces but not to those that lack surface GAGs. Collectively, these data show that given its expression in both the endospore and the exospore, EnP1 is a microsporidian cell wall protein that may function both in a structural capacity and in modulating in vitro host cell adherence and infection.
Eukaryotic Cell 09/2007; 6(8):1354-62. · 3.60 Impact Factor
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ABSTRACT: The infection process of intracellular opportunistic microsporidia involves the forcible eversion of a coiled hollow polar filament that pierces the host cell membrane, allowing the passage of infectious sporoplasm into the host cell cytoplasm. Although the exact mechanism of spore activation leading to polar filament discharge is unknown, we have shown that spore adherence to host cells, which is mediated by sulfated glycosaminoglycans, may play a vital role. When adherence is inhibited, host cell infection decreases, indicating a direct link between adherence and infection. The goal of this study was to evaluate the effects of exogenous divalent cations on microsporidia spore adherence and infection. Data generated using an in vitro spore adherence assay show that spore adherence is augmented by manganese (Mn2+) and magnesium (Mg2+), but not by calcium (Ca2+). However, each of the three divalent cations contributed to increased host cell infection when included in the assay. Finally, we show that Mn2+ and Mg2+ may activate a constituent on the microsporidia spore, not on the host cell, leading to higher infection efficiency. This report further supports recent evidence that spore adherence to the host cell surface is an important aspect of the microsporidial infection process.
FEMS Microbiology Letters 08/2006; 260(2):143-9. · 2.04 Impact Factor
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Journal of Eukaryotic Microbiology 02/2006; 53 Suppl 1:S68-9. · 2.66 Impact Factor
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ABSTRACT: Microsporidia are obligate intracellular opportunistic protists that infect a wide variety of animals, including humans, via environmentally resistant spores. Infection requires that spores be in close proximity to host cells so that the hollow polar tube can pierce the cell membrane and inject the spore contents into the cell cytoplasm. Like other eukaryotic microbes, microsporidia may use specific mechanisms for adherence in order to achieve target cell proximity and increase the likelihood of successful infection. Our data show that Encephalitozoon intestinalis exploits sulfated glycans such as the cell surface glycosaminoglycans (GAGs) in selection of and attachment to host cells. When exogenous sulfated glycans are used as inhibitors in spore adherence assays, E. intestinalis spore adherence is reduced by as much as 88%. However, there is no inhibition when nonsulfated glycans are used, suggesting that E. intestinalis spores utilize sulfated host cell glycans in adherence. These studies were confirmed by exposure of host cells to xylopyranoside, which limits host cell surface GAGs, and sodium chlorate, which decreases surface sulfation. Spore adherence studies with CHO mutant cell lines that are deficient in either surface GAGs or surface heparan sulfate also confirmed the necessity of sulfated glycans. Furthermore, when spore adherence is inhibited, host cell infection is reduced, indicating a direct association between spore adherence and infectivity. These data show that E. intestinalis specifically adheres to target cells by way of sulfated host cell surface GAGs and that this mechanism serves to enhance infectivity.
Infection and Immunity 03/2005; 73(2):841-8. · 4.16 Impact Factor
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ABSTRACT: Normal lymphoid tissue development and function depend upon chemokine-directed cell migration. Since chemokines signal through heterotrimeric G-protein-coupled receptors, RGS proteins, which act as GTPase-activating proteins for Galpha subunits, likely fine tune the cellular responses to chemokines. Here we show that Rgs1(-/-) mice possess B cells that respond excessively and desensitize improperly to the chemokines CXCL12 and CXCL13. Many of the B-cell follicles in the spleens of Rgs1(-/-) mice have germinal centers even in the absence of immune stimulation. Furthermore, immunization of these mice leads to exaggerated germinal center formation; partial disruption of the normal architecture of the spleen and Peyer's patches; and abnormal trafficking of immunoglobulin-secreting cells. These results reveal the importance of a regulatory mechanism that limits and desensitizes chemokine receptor signaling.
Molecular and Cellular Biology 08/2004; 24(13):5767-75. · 5.53 Impact Factor
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ABSTRACT: Giardia lamblia, a worldwide cause of diarrhoea, must differentiate into environmentally resistant cysts for dissemination and completion of its life cycle. Although G. lamblia is an early diverging eukaryote, encystation involves many complex cellular changes including formation of the cyst wall that contains at least two cyst wall proteins, cyst wall proteins 1 and 2. Cwp genes are transcribed only during encystation. In this study, we examine the regulatory elements for the encystation-specific gene cwp2. The 64 bp immediately upstream of the cwp2 open reading frame (-64 to -1 relative to ATG) was shown to be sufficient for the encystation-specific expression of luciferase. To determine which region(s) within this 64 bp contributed to encystation-specific expression in vivo, a series of deletions were cloned into a Giardia luciferase expression vector and their ability to control encystation-specific expression of luciferase was assessed. Deletion of elements in the -64 to -23 region of the cwp2 promoter significantly increased expression of luciferase in vegetative trophozoites, suggesting that this area contains a negative cis-acting element. Deletions of elements from -23 to -10 led to decreased expression in encysting cells, suggesting that this region may contain positive cis-acting elements. When the A/T-rich initiator was deleted but the cis-acting elements (-64 to -10) were retained, encystation-specific expression of luciferase was maintained but an aberrant transcriptional start site was utilised. These results indicate that Giardia has developed a classic repressor mechanism(s) that allows tight, encystation-specific control by the cwp2 promoter.
International Journal for Parasitology 10/2003; 33(10):1005-12. · 3.39 Impact Factor
